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The Effect of Formalin Preservation Time and Temperature on the Material Properties of Bovine Femoral Cortical Bone Tissue

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Abstract

Literature has reported controversial findings on whether formalin affected bone properties, or not, especially when different preservation time durations and temperatures were involved. Hence, accurately and systematically quantifying the effect of formalin on the mechanical properties of bone using a large dataset is crucial for assessing biomechanical responses based on fixed specimens. A total of 154 longitudinal and 149 transverse cuboid-shaped (12 mm × 2 mm × 0.5 mm) specimens from the midsection of 12 bovine femora from six bovines were prepared and assigned to ten groups, including fresh-frozen, formalin-preserved at 25 °C for 4 weeks and 8 weeks, and formalin-preserved at 4 °C for 4 weeks and 8 weeks. All specimens underwent quasi-static three-point bending tests with a loading rate of 0.02 mm/s. The Young’s modulus, yield stress, yield strain, tangent modulus, effective plastic strain, ultimate stress, and toughness were calculated by optimizing the material parameters to make the force–displacement curve of the finite element prediction consistent with the experimental curve, combined with specimen-specific finite element models. Preservation time and temperature both had significant effects on the Young’s modulus, yield stress, effective plastic strain, yield strain and ultimate stress of cortical bone (p < 0.05). The Young’s modulus, yield stress, and ultimate stress of longitudinal specimens decreased significantly with the increase of preservation time, and the yield strain increased significantly. As the preservation temperature increases, the Young’s modulus of the transverse sample decreased significantly, and the yield strain increased significantly. The preservation time mainly affects the longitudinal specimens, while the preservation temperature mainly affects the transverse specimens. Formalin preservation of bovine femoral cortical bones at a lower temperature and less than 4 weeks is recommended for biomechanical testing.

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Acknowledgments

This research was funded by the National Natural Science Foundation of China (51205118 and 11402296) and the State Key Laboratory of Advanced Design and Manufacturing for Vehicle Body, Hunan University (51475002). We also acknowledge support from the Canada Research Chairs program.

Conflict of interest

There are no conflicts of interest.

Author information

Authors and Affiliations

  1. State Key Laboratory of Advanced Design and Manufacturing for Vehicle Body, Hunan University, Changsha, 410082, China

    Guanjun Zhang, Shujing Wang, Songyang Xu & Zhonghao Bai

  2. Science and Technology on Integrated Logistics Support Laboratory, National University of Defense Technology, Changsha, 410073, China

    Fengjiao Guan

  3. Department of Mechanical and Materials Engineering, Faculty of Engineering, School of Biomedical Engineering, Western University, London, ON, N6A 5B9, Canada

    Haojie Mao

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  1. Guanjun Zhang
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Correspondence to Haojie Mao.

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Appendices

Appendix A: Grouping of the Five Preservation Methods for Longitudinal and Transverse Specimens

Groups

Specimen direction

Preservation temperature (°C)

Preservation time (weeks)

Sample size

Femur #1

Femur #2

Femur #3

Femur #4

Femur #5

Femur #6

Femur #7

Femur #8

Femur #9

Femur #10

Femur #11

Femur #12

Total

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

A/P/L/M

Total

LF

Longitudinal

–

–

1/1/1/0

1/1/1/1

1/1/1/1

1/1/1/1

1/1/1/1

1/1/0/1

1/1/1/1

1/0/1/1

1/0/1/1

0/1/1/1

1/1/1/1

1/1/1/1

11/10/11/11

43

LE4w-25

Longitudinal

25

4

1/0/1/1

0/1/1/1

1/1/1/0

1/0/1/1

1/1/1/0

1/0/1/1

1/1/0/1

1/1/0/1

1/1/1/1

1/1/1/0

1/0/1/1

0/1/1/1

10/9/9/9

37

LE8w-25

Longitudinal

25

8

1/1/0/1

1/1/0/1

1/1/1/1

0/1/1/1

1/0/1/1

1/1/1/0

1/1/1/0

0/1/1/1

1/1/0/1

1/0/1/1

0/1/1/1

1/0/1/1

9/9/9/10

37

LE4w-4

Longitudinal

4

4

0/1/1/0

0/1/0/0

0/0/1/0

1/0/0/0

0/1/0/1

0/0/0/1

0/0/0/1

0/1/1/0

0/0/1/0

1/0/0/0

1/0/0/1

1/0/0/0

4/4/4/4

16

LE8w-4

Longitudinal

4

8

1/0/0/1

1/0/1/0

0/0/0/1

0/1/0/1

0/0/0/1

0/1/1/0

0/1/1/0

1/0/0/0

1/1/0/0

0/1/0/1

1/0/1/0

0/1/1/0

5/6/5/5

21

TF

Transverse

–

–

1/1/1/1

0/1/1/1

1/1/1/1

1/1/1/1

1/1/1/1

1/1/1/1

1/0/1/1

1/1/1/1

1/1/1/1

1/1/1/0

0/1/1/1

1/1/1/0

10/11/12/10

43

TE4w-25

Transverse

25

4

0/1/1/1

1/1/1/0

1/1/1/1

1/1/1/1

1/0/1/1

1/1/1/1

1/1/1/1

1/1/1/1

0/1/1/1

1/0/1/1

1/1/1/1

1/1/0/1

10/10/11/11

42

TE8w-25

Transverse

25

8

1/1/1/0

1/1/1/1

0/1/1/1

1/1/1/0

1/1/1/1

0/1/1/1

1/1/1/1

1/0/1/1

1/1/0/0

1/1/1/1

1/0/0/1

1/0/1/1

10/9/10/9

38

TE4w-4

Transverse

4

4

1/0/0/0

0/0/0/1

0/1/0/0

0/0/0/1

1/0/0/0

0/0/1/0

0/0/1/0

0/1/0/0

0/0/1/0

0/0/0/1

1/0/0/0

0/1/0/0

3/3/3/3

12

TE8w-4

Transverse

4

8

0/0/0/1

1/0/0/0

1/0/0/0

0/0/1/0

0/1/0/0

1/0/0/0

0/1/0/0

0/1/0/0

1/0/0/1

0/1/0/0

0/0/1/0

0/0/1/1

4/4/3/3

14

Appendix B: Two-Way Analysis of Variance and Its Results

There are two factors to be concerned about in this study, preservation time and preservation temperature. To make the problem easier to analyze, one factor is fixed at a certain level (as a constant), and another factor is used as a variable for statistical analysis. Longitudinal specimens and transvers specimens were studied separately. For longitudinal specimens, the sets of LF/LE4w-25/LE8w-25 and LF/LE4w-4/LE8w-4 were used to investigate whether the preservation time had an effect on the mechanical properties of bone at 25 °C and 4 °C, respectively; the sets of LF/LE4w-4/LE4w-25 and LF/LE8w-4/LE8w-25 were used to investigate whether the preservation temperature had an effect on the mechanical properties of bone after preserved for 4 weeks and 8 weeks, respectively. The transverse specimens were also subjected to similar sets between groups (Table 3). For material parameters that satisfy a normal distribution with homogeneous variance, one-way analysis of variance (ANOVA) was used to study the significance of the differences in the mean of material parameters of each group in the set; otherwise, a nonparametric Kruskal–Wallis test was used. Multiple comparison method was used to study the significance of differences between groups in each of the above sets. Tukey’s test was used for the parameters that met homogeneity of variance; otherwise, Dunnett’s T3 test was used. All statistical analyses were performed by using SPSS (Version 21, IBM Corporation, Somers, NY, USA) with a significance level of 0.05 (Fig. 5, Tables 3, 4).

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Zhang, G., Wang, S., Xu, S. et al. The Effect of Formalin Preservation Time and Temperature on the Material Properties of Bovine Femoral Cortical Bone Tissue. Ann Biomed Eng 47, 937–952 (2019). https://doi.org/10.1007/s10439-019-02197-1

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